This invention relates to solid sorbents having surface defining sites capable of selectively binding a preselected macromolecule, useful in the separation of a target solute from a complex mixture and in various types of analyses. The invention also relates to a family of synthetic techniques useful in fabricating such surfaces.
Adsorption of macromolecules such as proteins to surfaces involves attraction at multiple sites through hydrophobic, electrostatic, and hydrogen bonding. Surfaces used in chromatographic packing materials therefore have a high density of ionic, hydrophobic or hydroxyl containing groups available for this adsorption process. The interface between the surface and adsorbed proteins may cover between about 10-100 surface groups on the sorbent, depending on the surface density of the charged or other groups and on the size of the protein. Adsorption typically occurs through 5 to 10 groups on the surface of the protein, so there is a large excess of surface functional groups. As the surface density of functional groups on a sorbent decreases, the strength of protein adsorption typically decreases rapidly. Although the number of groups on the sorbent surface is more than adequate for binding, the groups are not distributed properly in space.
The effect is illustrated schematically in FIGS. 1A and 1B. In FIG. 1A, the accessible surface area of a protein, depicted at 10, has five dispersed anion groups, all of which lie close to one or more cation groups disposed at high density in a field on the surface 12 of the adsorbent. As shown in FIG. 1B, at lower surface density, the protein will be less avidly bound, as the spatial distribution of the anions on the protein surface do not match up well with the positioning of the cations on the sorbent.
Of course, real behavior differs in several respects from the oversimplified situation depicted, as, for example, 1) charged groups are randomly positioned on the sorbent, 2) adsorption occurs in three dimensions, e.g., the charge pair in the square shown in FIG. 1B may be spaced apart in a direction normal to the plane of the paper, 3) the protein may have cation groups on its surface which will be repelled by the cation surface and 4) there are other physical interactions at work in addition to electrostatic attraction.
This complimentary adsorption phenomenon is used most widely in chromatographic processes involving purification and analysis of analytes exploiting differential sorption properties of solutes in a mixed solution. Those who manufacture chromatographic systems generally seek to make the surface of the sorbent as homogeneous as possible, and to have a high density of functional groups. Complementarity is based on the presence of a single set of functional groups on the sorbent surface being complementary with a subset of the functional groups on the analyte. In adsorption chromatography, for example, silanol groups at the surface of silica are used to associate with solutes through hydrogen bonding. This generally is achieved in an organic solvent where hydrogen bonding is strong. In ion exchange chromatography, as noted above, a charged surface interacts with a molecular species of opposite charge through electrostatic interaction. The driving force for interaction is based in part on enthalpic changes upon binding and in part upon entropic effects from the displacement of water at the surface of both the sorbent and the sorbate. In reversed-phase and hydrophobic interaction chromatography, the entropic effect is exploited to its fullest as hydrophobic molecules are forced against the sorbent surface to minimize their hydrophobic contact area with the relatively polar solvent. Immobilized metal affinity chromatography is yet another example of the participation of complementary functional groups in the adsorption process. In this system, immobilized metal coordination compounds interact in the presence of metal such as zinc or copper with histidine on an accessible exterior surface of a polypeptide. This association causes the differential adsorption of polypeptides based on number and spatial arrangement of histidines. All of these systems exploit a surface having a random high ligand density. No attempt is made to match specific structural features of the molecule with structural features of the sorbent surface.
Affinity chromatography is based on exploitation of biological systems to achieve intermolecular docking and adsorption. In this system, the surface of the sorbent is caused to mimic a biological substance which naturally associates with a polypeptide. Affinity interactions generally are based on multiple phenomenon including electrostatic attraction, hydrophobic interaction, hydrogen bonding, and stereochemical interfit.
Reversible binding interactions between pairs of biological macromolecules such as ligands and receptors or antibodies and antigens have been exploited widely to construct systems taking advantage of the exquisite specificity and affinity of these interactions. Affinity chromatography often involves the immobilization of specific binding protein, previously typically polyclonal antisera, but now commonly monoclonal antibody, to a high surface area solid matrix such as a porous particulate material packed in a column. The feed mixture is passed through the column where the target solute binds to the immobilized binding protein. The column then is washed and the target substance subsequently eluted to produce a fraction of higher purity. Solid material comprising such specific binding surfaces also are used in immunoassay where immobilized binding protein is used to capture selectively and thereby separate an analyte in a sample.
There has been steady, sometimes dramatic improvement in methods for producing specific binding protein useful in such contexts and for immobilizing them on surfaces. Thus, monoclonal antibodies largely replaced polyclonal antisera obviating the need to purify the antibodies from bleedings, enabling epitope-specific binding, and established a technology capable theoretically of producing industrial quantities of these valuable compounds. More recently, advances in protein engineering and recombinant expression have permitted the design and manufacture of totally synthetic binding sites mimicking the antigen binding domains of the natural antibodies.
While this technology is very useful it is not without its drawbacks. The binding proteins are high molecular weight biological macromolecules whose function depend on maintenance of a tertiary structure easily altered upon exposure to relatively mild condition in use or storage. Furthermore, while it is now within the skill of the art to prepare antibodies or their biosynthetic analogs having specificity for a predetermined target molecule, the preparative technique are time-consuming and costly, purification is difficult, and the techniques for immobilizing them onto surfaces at high density while maintaining activity is imperfect. Furthermore, when such specific binding surfaces are used for the purification of substances intended for therapeutic or prophylactic use in vivo, they introduce a risk of contamination of the product by foreign biological material. This complicates quality control, increases the complexity of the design of a purification system, and increases the expense and time required to obtain regulatory approval of the drug.
Molecular recognition is an important phenomenon in biological systems. The area involved in the interface between the surface and the analyte can be as small as 10 to 100 square .ANG. in the case of amino acids and monosacharides and range to as large as thousands of .ANG. in the interface between polypeptides forming quaternary structure. At the level between about 10-100 square .ANG. surface area in the interface, man has been successful in mimicking nature. This is the basis for modern affinity chromatography discussed above. However, the ability to discriminate could be increased by using a broader surface area at the interface.
It is an object of this invention to provide rationally designed, stable, inexpensive to manufacture surfaces on solid materials comprising a multiplicity of site which reversibly, noncovalently bind with high specificity and affinity a preselected target molecule. Another object is to provide such materials adapted for use in various types of analyses involving specific binding which heretofore have been limited to the use of immobilized macromolecules of biological origin. Still another object is to provide solids having surfaces containing specific binding sites useful for both preparative and analytical chromatographic separations, which, as compared with conventional affinity chromatography surfaces, are more durable, useful over a greater range of conditions, and less expensive to manufacture. Still another object is to provide a family of synthetic techniques which permit synthesis of rationally designed surfaces containing a multiplicity of regions which, through a combination of spatially matched electrostatic attraction, hydrophobic interaction, chelation, hydrogen bonding, and/or stereochemical interfit, are capable of binding to any given macromolecular surface.
These and other objects and features of the invention will be apparent from the drawing, description, and claims which follow.